Cells, Reagents and Culture
The Bacillus cereus strain (RSVF1 strain 4342), Bacillus cereus ATCC 11778, Bacillus cereus ATCC10876, and Bacillus thuringiensis ATCC 10792 were procured from the American Type Culture Collection (ATCC, Manassas, VA). Animal vaccine strain of B. anthracis (Sterne 34F2) originally obtained from Colorado Serum Co. (Denver, CO) was a generous gift from Dr. Duncan, CBER, FDA. We utilized brain-heart infusion (BHI) broth (Becton Dickinson, Sparks, MD) for culturing this strain and Miller's Luria Bertani (LB) broth (Mediatech Inc, Herndon, VA) for others. All bacterial cultures were grown at 37°C.
Synthesis of PlyG peptides
Six varying lengths of peptides (named PlyG-P1 through PlyG-P6) and ranging from 10 to 20 residues, representing amino acid position 185 to 204 encompassing the cell wall binding essential domain of wild type PlyG and its mutant forms were synthesized. The amino acid sequences of the peptides were based on the previously published amino acid sequence of PlyG C-terminal region . The peptides were synthesized at our Core Facility in CBER, FDA, biotinylated with a C6-linker and purified by High Pressure Liquid Chromatography (HPLC) for detection using a horse radish peroxidase (HRP)-conjugate (Figure 1). Peptides were reconstituted in 100 mM NaPO4 and 150 mM NaCl, pH 7.2 buffer at room temperature to a final concentration of 1 M. This stock solution was diluted as needed for further use. We confirmed that all six peptides to have the biotin linkers and are recognized by steptavidin-HRP conjugate, prior to initiating the binding assays.
Dot blot assay
5-ml early-log-phase culture of B. cereus-4342, B. cereus ATCC 11778, B. cereus ATCC 10876, and B. thuringiensis ATCC 10792 were grown in LB broth and B. anthracis-Sterne was grown in BHI broth for 3 hr followed by centrifugation at 3,000 × g. The cell pellet was resuspended and 10-fold serial dilutions were made in 1× PBS (pH 7.4, Mediatech Inc, Herndon, VA). Bacterial suspension (103 CFU/ml) was spotted directly onto a nitrocellulose membrane essentially as described . The membrane was incubated in 5% bovine serum albumin (BSA) solution (Sigma, St. Louis, MO) for 2 h to eliminate non-specific signals on the membrane. Each membrane was incubated separately with one peptide at a final concentration of 1.5 mM. Subsequently the membranes were washed three times with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20 (Aldrich Chemical Company, Milwaukee, WI). The membranes were then incubated with 1:10,000 diluted streptavidin-HRP conjugate (Upstate, Temecula, CA) for 60 min at RT and washed three times with TBST buffer. Following washing, the membrane was developed using a DAB (3, 3'- diaminobenzidine) substrate kit (Zymed Laboratories, Carlsbad, CA). The development of colored spots in locations where the bacteria were spotted on the membrane was inferred as positive for peptide binding. The membranes were photographed for archiving the data.
Enzyme-Linked ImmunoSorbent Assay (ELISA)
The assay was carried out as described previously  with modifications. Briefly, 5-ml of early-log-phase bacterial cultures of B. cereus-4342, B. anthracis-Sterne, B. cereus-11778, B. cereus-10876, and B. thuringiensis were centrifuged at 3,000 × g. The cell pellet was resuspended and 10-fold serial dilutions were made in 1× PBS. Bacterial cell suspension at a concentration of 103 CFU was then layered into the wells of 96-well micro plate (Becton Dickinson, Bedford, MA) and incubated overnight at RT. Subsequently, cells were fixed in ethanol (Aldrich Chemical Company, Milwaukee, WI) for 10 min and the plates were air-dried. Wells were blocked with 5% BSA (Sigma, St. Louis, MO) for 60 min at RT, rinsed with PBS and then PlyG peptides suspended in PBS at a final concentration of 10 mM were added to all the wells and incubated for 15 min. Following incubation, wells were washed 3 times with PBST buffer (PBS pH 7.4, 0.01% Tween 20). The wells were further incubated with 1:10,000 dilution of streptavidin-HRP conjugate (Upstate, Temecula, CA) for 15 min and washed with PBST buffer. Tetramethylbenzidine (TMB) membrane peroxidase substrate (Zymed Laboratories, Carlsbad, CA) system was used to detect the enzyme label in accordance with the manufacturer's instructions. The color development in the 96-well plate was recorded by using Synergy 4™ BioTek micro plate reader (BioTek Instruments, Winooski, VT) at 490 nm wavelength. Assays were repeated three times for statistical analysis.
Q-dot based fluorescence assay
Log-phase cultures of B. cereus-4342, B. anthracis-Sterne, B. cereus-11778, B. cereus-10876, and B. thuringiensis were subjected to low-speed centrifugation and the pellet was resuspended in 1 × PBS. Ten-fold serial dilutions were each made in 1 ml of human plasma  and 103 CFU/ml was used for spiking the plasma. The rationale for spiking 103 CFU/ml of bacteria was based on the consensus in the field, which suggests that the limit of detection for commercially available rapid but less sensitive bacterial detection systems is 103–105 CFU/ml (http://www.veraxbiomedical.com). Peptide binding and detection assay was performed in a final volume of 100 μl in an eppendorf tube kept at RT for 90 min. Following the binding step, tubes were centrifuged for 5 minutes at 3,000 × g and the pellets were washed with PBS and resuspended in 100 μl of 1:10,000 diluted streptavidin-conjugated Q dots (QD 605) solution (Invitrogen, Gaithersberg, MD). After incubating for 90 min at RT, samples were centrifuged again as above and the pellets were resuspended in 150 μl of PBS. Samples were then placed on a glass slide (Mercedes Medical, Sarasota, FL) or a micro well plate (Becton Dickinson Lab ware, Bedford, MA) and analyzed either under a fluorescence microscope  using Nikon Eclipse TE2000-U (Nikon Instruments, Melville, NY) or by fluorometry  using Synergy 4™ BioTek micro plate reader (BioTek Instruments, Winooski, VT) respectively. Images as visualized under the microscope were captured by an intensified cooled charge-coupled device-equipped camera (Nikon Eclipse TE2000-U).
We performed all assays described here in triplicates. Mean values ± SD (Standard Deviation) were calculated using Microsoft Excel®. Statistical analyses were performed using Student's t test and values were considered significant when P< 0.05.